Complete Structure Elucidation of Shishididemniols, Complex Lipids

Complete Structure Elucidation of Shishididemniols, Complex. Lipids with Tyramine-Derived Tether and Two Serinol Units, from a Marine Tunicate of the ...
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Complete Structure Elucidation of Shishididemniols, Complex Lipids with Tyramine-Derived Tether and Two Serinol Units, from a Marine Tunicate of the Family Didemnidae Hirotsugu Kobayashi,† Jun’ichiro Ohashi,† Tsuyoshi Fujita,‡ Takashi Iwashita,‡ Yoichi Nakao,† Shigeki Matsunaga,*,† and Nobuhiro Fusetani† Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, and Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan [email protected] ReceiVed September 29, 2006

Two new serinolipid derivatives, shishididemniols A (1) and B (2), were isolated as antibacterial constituents of a tunicate of the family Didemnidae. The structure of 1 was elucidated by interpretation of spectral data and the application of the modified Mosher method to 1 and its suitable degradation products. Compound 2 was the chlorohydrin of 1. Compounds 1 and 2 exhibited antibacterial activity against fish pathogenic bacterium Vibrio anguillarum.

Introduction

This paper describes the isolation, structure elucidation and antimicrobial activitiy of these compounds.

Fish and shellfish diseases are the most serious concerns for aquaculture today.1 The popular action taken in fish farms is chemotherapy using chemical substances such as antibiotics and synthetic organic compounds to control these diseases.1 However these chemical substances pose environmental and hygienic problems.1 We have been searching for antimicrobial metabolites against fish pathogenic bacteria from marine natural products that are expected to be environmentally benign. As a part of the study, we have discovered novel bromotyrosine derivatives from the marine sponge Hexadella sp.2 that exhibited antibacterial activity against the fish pathogenic bacterium Aeromonas hydrophila. Subsequently, we found that the extract of a tunicate of the family Didemnidae collected in southern Japan exhibited activity against the bacterium Vibrio anguillarum, which causes vibriosis in fish.3 Bioassay-guided fractionation afforded two novel serinolipid derivatives, shishididemniols A (1) and B (2). Results and Discussion †

University of Tokyo. ‡ Suntory Institute.

(1) Shao, Z. J. AdV. Drug. DeliV. ReV. 2001, 50, 229-243. (2) Matsunaga, S.; Kobayashi, H.; van Soest, R. W. M.; Fusetani, N. J. Org. Chem. 2005, 70, 1893-1896. (3) Roberts, R. J. In Bacterial Disease of Fish; Inglis, V., Roberts, R. J., Bromage, N. R., Eds.; Blackwell Science: Oxford, 1993; pp 109-121.

The EtOH extract of the tunicates (780 g) was partitioned between CHCl3 and H2O, and the aqueous layer was further extracted with n-BuOH. The n-BuOH fraction was separated by ODS flash chromatography followed by reversed-phase HPLC to afford shishididemniol A (1) and B (2) in yields of 10.1021/jo062013m CCC: $37.00 © 2007 American Chemical Society

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Structure Elucidation of Shishididemniols

395 mg (5.1 × 10-2 % wet weight) and 152 mg (1.9 × 10-2 % wet weight), respectively. The molecular formula of shishididemniol A (1) was established as C45H81N3O11 on the basis of NMR and HRESIMS data. Interpretation of the 1H NMR spectrum (in DMSO-d6) together with HSQC data showed the presence of an Nsubstituted methylene (δH 3.12 and 3.31, δC 46.2), two nitrogenous methines (δH 3.23, δC 52.0; δH 4.02, δC 50.2), five oxygenated methylenes (δH 3.40 (2H), δC 70.7; δH 3.44 and 3.50, δC 67.5; δH 3.48 and 3.56, δC 58.7; δH 3.93 and 3.95, δC 66.5; δH 3.46 and 3.49, δC 60.1), four oxymethines (δH 3.80, δC 70.8; δH 3.19, δC 68.7; δH 3.33, δC 69.5; δH 4.55, δC 70.8), two epoxide methines (δH 2.69, δC 60.5; δH 2.87, δC 56.2), a triplet methyl (δH 0.97, δC 9.90), a methylene (δH 2.10, δC 28.4), two aromatic signals [δH 6.89 (2H), δC 114.1 (2C); δH 7.22 (2H), δC 127.1 (2C)], two amide protons (δ 7.51 and 7.74), and seven exchangeable protons (δ 4.19, 4.83, 4.85, 5.24, 5.41, 5.41, and 7.80). The remainder of the 1H NMR signals were attributed to long methylene chains (δ 1.15-1.45). In addition, two non-protonated sp2 carbon signals (δ 135.6 and δC 157.7) and two carbonyl carbon signals (δ 173.0 and 173.9) were observed in the 13C NMR spectrum (Table 1). Interpretation of 2D NMR data led to substructures a-c (Figure 1A). In substructure a, the propionamide moiety (C12′ to C-14′) was identified from the COSY and HMBC data. The COSY spectrum revealed spin systems for C-9′-C10′(-NH10′)-C-11′-OH-11′ and NH-1′-C-1′-C-2′-OH. The connectivity between N-10′ and C-12′ was determined by HMBC crosspeaks, NH-10′/C-12′ and H-10′/C-12′. 1H and 13C NMR chemical shift values clearly indicated the presence of a parasubstituted phenyl ether moiety. The ether linkage was established by the HMBC cross-peak, H-9′/C-6′, while further HMBC correlations, H-2′/C-3′, H-2′/C-4′(8′), and H-4′(8′)/C-2′, demonstrated the connectivity between C-2′ and C-3′. The connectivities from OH-2 to H2-4 and from OH-6 to H2-9 were assigned on the basis of the COSY data. Further analysis of the COSY and TOCSY data indicated that the C-6 oxymethine proton was adjacent to H2-5, which was in turn correlated with H2-4. The 1H-1H coupling constant between H-7 and H-8 (J ) 4.1 Hz) revealed that C-7 and C-8 were a part of a cis-epoxide. HMBC cross-peaks, NH-1′/C-1, H-2/C-1, and OH-2/C-1, showed the connectivity between C-1 and N-1′. Substructure b comprises the terminus of an alkyl chain attached to a serinol ether.4,5 The serinol unit (O-C-29-C-30C-31-OH-31) was clearly defined by the COSY and HSQC data. The chemical shift value of C-30 at δC 52.0 and the ninhydrinpositive property of 1 implied that C-30 was substituted by a free amino group. The ether linkage was established by HMBC cross-peaks from H2-28 to C-29. Substructure c contains an oxymethine group placed in the middle of a long methylene chain. The COSY spectrum showed that C-16 oxymethine proton was coupled to OH-16 and two pairs of methylene protons (H2-15 and H2-17). Therefore, substructure c should be inserted between substructure a and b via methylene chains. The location of substructure c in the alkyl chain was determined by analysis of FAB-MS/MS data of 1. Notable fragment ions observed at m/z 258 and 288 allowed us to locate the hydroxyl group at C-16 (Figure 1B). (4) Gonza´lez, N.; Rodriguez, J.; Jime´nez, C. J. Org. Chem. 1999, 64, 5705-5707. (5) Mitchell, S. S.; Rhodes, D.; Bushman, F. D.; Faulkner, D. J. Org. Lett. 2000, 2, 1605-1607.

1H and B (2) in DMSO-d6a

TABLE 1.

13C

NMR Data for Shishididemniols A (1) and 1

position 1 2 3a 3b 4a 4b 5a 5b 6 7 8 9a 9b 10-14 15 16 17 18-25 26 27 28 29a 29b 30 31a 31b 1′a 1′b 2′ 3′ 4′, 8′ 5′, 7′ 6′ 9′a 9′b 10′ 11′a 11′b 12′ 13′ 14′ OH-2 OH-6 OH-7 OH-16 NH2-30 OH-31 NH-1′ OH-2′ NH-10′ OH-11′

δH (mult, J Hz) 3.80 (br) 1.42 (m) 1.55 (m) 1.31 (m) 1.42 (m) 1.29 (m) 1.40 (m) 3.19 (br) 2.69 (dd, 4.1, 8.2) 2.87 (ddd, 4.1, 4.1, 8.2) 1.28 (m) 1.54 (m) 1.15-1.45 (m) 1.21-1.34 (m) 3.33 (m) 1.21-1.34 (m) 1.15-1.45 (m) 1.27 (m) 1.49 (tt, 7.6, 7.6) 3.40 (t, 7.6) 3.44 (m) 3.50 (dd, -10.5, 4.8) 3.23 (m) 3.48 (m) 3.56 (m) 3.12 (m) 3.31 (m) 4.55 (br)

3.93 (dd, -9.9, 5.9) 3.95 (dd, -9.9, 5.7) 4.02 (m) 3.46 (m) 3.49 (m)

4.19 (br) 7.80 (br) 5.24 (br) 7.51 (t, 5.9) 5.41 (d, 4.1) 7.74 (d, 7.8) 4.83 (br)

173.9 (C) 70.8 (CH) 34.3 (CH2) 20.4 (CH2) 33.7 (CH2) 68.7 (CH) 60.5 (CH) 56.2 (CH) 27.9 (CH2) 28.9-29.3 37.2 (CH2) 69.5 (CH) 37.2 (CH2) 28.9-29.3 25.5 (CH2) 28.9 (CH2)

δH (mult, J Hz) 3.81 (br) 1.43 (m) 1.57 (m) 1.37 (m) 1.44 (m) 1.31 (m) 1.38 (m) 3.46 (m) 3.27 (t, 4.4) 3.99 (dt, 8.8, 4.4) 1.67 (m) 1.77 (m) 1.17-1.31 (m) 1.22-1.34 (m) 3.33 (m) 1.22-1.34 (m) 1.17-1.31 (m) 1.27 (m) 1.49 (m)

δC (multb) 173.9 (C) 70.9 (CH) 34.4 (CH2) 21.0 (CH2) 32.6 (CH2) 70.9 (CH) 75.6 (CH) 66.2 (CH) 34.4 (CH2) 29.4-29.8 37.7 (CH2) 69.5 (CH) 37.7 (CH2) 29.4-29.8 25.5 (CH2) 28.6 (CH2)

70.7 (CH2) 67.5 (CH2)

3.39 (m) 3.43 (m) 3.49 (m)

70.7 (CH2) 67.7 (CH2)

52.0 (CH) 58.7 (CH2)

3.20 (m) 3.48 (m) 3.55 (dd, 4.7, 10.9) 3.13 (m) 3.31 (m) 4.55 (br)

52.0 (CH) 59.0 (CH2)

46.2 (CH2)

7.22 (d, 8.7) 6.89 (d, 8.7)

2.10 (q, 7.2) 0.97 (t, 7.2) 5.41 (d, 4.1) 4.85 (br)

2 δC (multb)

70.8 (CH) 135.6 (C) 127.1 (CH) 114.1 (CH) 157.7 (C) 66.5 (CH2)

50.2 (CH) 60.1 (CH2) 173.0 (C) 28.4 (CH2) 9.90 (CH3)

7.22 (d, 8.8) 6.89 (d, 8.8) 3.93 (dd, -9.8, 6.0) 3.95 (dd, -9.8, 5.4) 4.03 (m) 3.46 (m) 3.50 (m) 2.10 (q, 7.7) 0.97 (t, 7.7) 5.48 (br) 4.19 (br) 4.82 (br) 4.19 (br) 7.61 (br) 4.83 (br) 7.50 (t, 5.8) 5.42 (br) 7.75 (d, 8.3) 4.82 (br)

46.1 (CH2) 70.9 (CH) 135.6 (C) 127.1 (CH) 114.1 (CH) 157.7 (C) 66.4 (CH2)

50.2 (CH) 60.1 (CH2) 173.1 (C) 28.4 (CH2) 9.91 (CH3)

a 600 MHz for 1H NMR and 150 MHz for 13C NMR. b Multiplicity from HSQC experiment.

The stereochemistry of C-6 with respected to that of C-7 in 1 could not be determined by analysis of NMR data.6,7 This (6) Sugiyama, T.; Yamashita, K. Agric. Biol. Chem. 1980, 44, 19831984. (7) Graham, S. M.; Prestwich, G. D. J. Org. Chem. 1994, 59, 29562966. (8) Righi, G.; Ronconi, S.; Bonini, C. Eur. J. Org. Chem. 2002, 15731577.

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FIGURE 1. (A) Partial structures of shishididemniol A (1). (B) FAB-MS/MS analysis of 1. SCHEME 1 a

a Reagents and conditions: (a) Cbz-Cl, Et N, CHCl /MeOH (3:1), rt, 16 h; (b) MgBr OEt Et O, rt, 3 h; (c) 2,2-dimethoxpropane, PPTS, CH Cl rt, 1 3 3 2 2, 2 2 2, h; (d) HCl/MeOH, 100 °C, 4 h; (e) hydrazine, 120 °C, 12 h.

was shown by converting 1 to the 6,7-O-isopropylidene derivatives 5, via the N-Cbz-protected bromohydrin 4 (Scheme 1).8 NOESY cross-peaks observed between one acetonide methyl signal (δH 1.40) and H-6 and between another methyl signal (δH 1.37) and H-7 showed the trans-relationship for H-6 and H-7. Therefore, the relative stereochemistry from C-6 to C-8 in 1 was assigned as threo-cis-2,3-epoxy alcohol (6S*,7R*,8S*). The absolute stereochemistry of C-2′ and C-10′ in 1 was envisaged to be determined by application of the modified 1220 J. Org. Chem., Vol. 72, No. 4, 2007

Mother’s method9,10 to compound 6, which was expected to be obtained by acidic hydrolysis of 1. We used (R)-2-amino-1phenylethanol, which was commercially available, and (S)-2amino-3-phenoxy-1-propanol (9) as model compounds to assign the stereochemistry of C-2′ and C-10′ in 6. Compound 9 was (9) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092-4096. (10) Seco, J. M.; Latypov, Sh. K.; Quin˜oa´, E.; Riguera, R. J. Org. Chem. 1997, 62, 7569-7574.

Structure Elucidation of Shishididemniols

FIGURE 2. 1H NMR chemical shift values for 10a/10b, 11a/11b, and 12a/12b. SCHEME 2 a

synthesized as outlined in Scheme 2. N-Boc-L-serine methyl ester was converted to a chiral alcohol 7 in two steps according to Garner’s procedure.12 Treatment of 7 with triphenylphosphine and diethyl azodicarboxylate at 80 °C in toluene with phenol provided the phenyl ether 8,13,14 which was deprotected with TFA to yield the expected compound 9. The (R)- and (S)-MTPA derivatives of (R)-2-amino-1-phenylethanol and those of compound 9 gave characteristic 1H NMR profiles, indicating that the stereochemistry at C-2′ and C-10′ in 6 could be determined by the modified Mosher method. Acidic methanolysis product of 1 was separated by ODS column chromatography to afford a fraction that contained UV-absorbing polar material. This material, which gave a single set of 1H NMR signals, was converted to the (R)- and (S)-MTPA derivatives. To our disappointment H2-1′ and H-2′ were doubled, implying that the benzyl carbon (C-2′) in 6 was racemized by acid and what we isolated was a 1:1 mixture of 6 and 6a. As a method to cleave amide bonds under a milder condition, we chose hydrazinolysis. Compound 1 was hydrazinolyzed11 to yield a fraction that contained 6, which was then converted to the (R)- and (S)MTPA derivatives (10a and 10b, respectively). Only one set of signals were observed in 1H NMR spectra of 10a and 10b.

The absolute stereochemistry of C-2′ and C-10′ in 6 was determined by comparison of 1H NMR spectra of 10a and 10b with those of 11a and 11b and those of (R)- and (S)-MTPA derivatives of (R)-2-amino-1-phenylethanol (12a and 12b, respectively) (Figure 2). 1H NMR chemical shift values of H21′ and H-2′ of 10a and 10b were in remarkable agreement with those of the corresponding signals of 12a and 12b, respectively, whereas the chemical shift values of H2-9′, H-10′, and H2-11′ agreed well with those of the corresponding signals of 12a and 12b, respectively. Therefore, the absolute stereochemistry of C-2′ and C-10′ was assigned as R and S, respectively (Figure 2). The absolute stereochemistry of C-2, C-6, and C-30 in 1 was determined by application of the modified Mosher method to 1 and 5. Treatment of 1 with (S)-MTPACl and (R)-MTPACl yielded the (R)-MTPA derivative (13a) and (S)-MTPA derivative (13b), respectively. Analysis of ∆δS-R values between 13a and 13b allowed the assignment of 6S and 30S (Figure 3). The negative ∆δS-R values observed for H2-3, H2-4, and H2-5 implied the 2S-stereochemistry. In order to exclude the effect of the C-6 MTPA ester, the acetonide 5 was converted to the (R)-MTPA derivative (14a) and (S)-MTPA derivative (14b). The negative ∆δS-R values observed for H2-3 to H2-8 signals confirmed the 2S-stereochemistry (Figure 3). Because C-16 was too far from the nearest functionalized carbon, it was not possible to detect the effects of MTPA esters on chemical shifts of assigned proton signals, e.g., H-8 and H29. This problem was hampered by esterification with 2NMA (2-naphthylmethoxyacetic acid), which was reported to exhibit anisotropic effects to more distant protons.15 In order to avoid confusion in assignments, we intended to introduce one 2NMA group to a derivative of 1. For that purpose the bromohydrin 4 was treated with NaIO4 followed by reduction with NaBH4 to obtain 15, whose two primary alcohols were protected with TBDPS groups16,17 to yield 16 (Scheme 3). The secondary alcohol in 16 was esterified with (R)-2NMA or (S)-2NMA in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

(11) Akabori, S.; Ohno, K.; Narita, K. Bull. Chem. Soc. Jap. 1952, 25, 214-218. (12) Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361-2364. (13) Mitsunobu, O. Synthesis 1981, 1-28. (14) Pave´, G.; Usse-Versluys, S.; Viaud-Massuard, M.; Guillaumet, G. Org. Lett. 2003, 5, 4253-4256.

(15) Kusumi, T.; Takahashi, H.; Hashimoto, T.; Kan, Y.; Asakawa, Y. Chem. Lett. 1994, 1093-1094. (16) Green, T. W.; Wuts, P. G. M. Protecting Groups in Organic Synthesis, 3rd ed.; Wiley-Interscience: New York, 1999. (17) Matsunaga, S.; Liu, P.; Celatka, C. A.; Panek, J. S.; Fusetani, N. J. Am. Chem. Soc. 1999, 121, 5605-5606.

a Reagents and conditions: (a) phenol, DEAD, PPh , toluene, 80 °C, 18 3 h; (b) CH2Cl2/TFA (1:1), rt, 2 h; (c) (S)-or (R)-MTPA-Cl, pyridine, rt, 30 min.

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FIGURE 3. (A) Distribution of ∆δS-R values for the MTPA derivatives 13a/13b. (B) Distribution of ∆δS-R values for the MTPA derivatives 14a/14b. (C) Distribution of the 2NMA esters of ∆δR-S values for 17a/17b.

SCHEME 3 a

a Reagents and conditions: (a) NaIO , MeOH/H O (4:1), rt, 1 h; then 4 2 NaBH4, rt, 15 min; (b) TBDPSCl, pyridine, rt, overnight; (c) (R)- or (S)2NMA, EDC, DMAP, DMAP‚Cl, CH2Cl2, rt, overnight.

hydrochloride (EDC), DMAP, and DMAP‚HCl18 in CH2Cl2 to give (R)- or (S)-2NMA derivatives (17a and 17b, respectively). Comparison of 1H NMR data of 17a and 17b showed that the absolute configuration at C-16 was R (Figure 3). Therefore, the stereochemistry of 1 was determined to be 2S,6S,7R,8S,16R,30S,2′R,10′S. Shishididemniol B (2) had a molecular formula of C45H82ClN3O11 as established by HRESIMS, which implied the presence of one less unsaturation equivalent than 1. The 1H and 13C NMR spectra of 2 were almost superimposable on those of 1 except for the replacement of the epoxide group by an oxymethine (δH 3.27, δC 75.6) and a chlorinated methine (δH 3.99, δC 66.2), suggesting that 2 was a HCl adduct of 1. Interpretation of NMR and FAB-MS/MS data of 2 confirmed that 2 was the 7-hydroxy-8-chloro-derivative of 1. In order to determine the stereochemical relationship between C-6 and C-7 in 2, its N-Cbz derivative 18 was converted to the acetonide 19 (Scheme 4). On the basis of NOESY correlations observed between one singlet methyl signal (δH 1.38) and H-6 and between another singlet methyl signal (δH 1.37) and H-7, the relative stereochemistry of C-6 and C-7 in 2 was assigned as identical with that in 1. Treatment of 18 with potassium carbonate19 in MeOH afforded the epoxide 3 ([R]20D -17.2 (c 0.10 MeOH)), which was indistinguishable from compound 3 derived from 1 ([R]20D -17.6 (c 0.10 MeOH)) in the [R]D and (18) Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394-2395. (19) Norman, B. H.; Hemsheidt, T.; Schults, R. M.; Andis, S. L. J. Org. Chem. 1998, 63, 5288-5294.

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1H

NMR. Therefore, the absolute stereochemistry of 2 was determined to be 2S,6S,7R,8R,16R,30S,2′R,10′S. Two classes of related serinolipid derivatives, didemniserinolipids4 and cyclodidemniserinol trisulfate,5 have been reported from tunicates of the genus Didemnum. The structure of didemniserinolipid B, whose serinol moiety was in the S configuration, was determined by total synthesis.20 Shishididemniols, didemniserinolipids, and cyclodidemniserinol trisulfate all possess C28-polyketide acid in the central part of the molecule. Unlike didemniserionlipids and cyclodidemniserinol trisulfate, shishididemniols A and B had two serinol and an oxygenated tyramine moieties and were not sulfated. Shishididemniols A and B showed antibacterial activity in disk agar diffusion assay against the fish pathogenic bacterium V. anguillarum (20 µg/ 6.5 mm φ disk zone of inhibition; 8 and 7 mm for 1 and 2, respectively). Experimental Section Animal Material. The tunicate was collected by hand using SCUBA at depths of 5-15 m off Kushizaki in Shishijima (32° 15′ N, 130° 14′ E) of the Amakusa Islands and was kept frozen at -20 °C until processed. The tunicate was identified as a member of the family Didemnidae on the basis of the presence of characteristic spicules.21 Extraction and Isolation. The frozen tunicates (780 g wet weight) were extracted four times with EtOH, and the combined extracts were concentrated and partitioned between water and CHCl3. The aqueous layer was further extracted with n-BuOH. The n-BuOH fraction was separated by ODS flash chromatography with aqueous MeOH. The fraction eluted with 70% MeOH was separated by ODS HPLC (72% MeOH containing 0.2 M NaClO4) to afford shishididemniols A (1, 395.4 mg, 5.1 × 10-2 % based on wet weight) and B (2, 151.6 mg, 1.9 × 10-2 % based on wet weight). Shishididemniol A (1). Colorless oil; [R]20.5D -33.7 (c 1.00, MeOH); UV (MeOH) λmax 275 nm ( 1195), 281 (1017); IR (film) νmax 3428, 1655 cm-1; HRESIMS m/z 840.59682 (M + H)+ (calcd for C45H82N3O11, ∆ +1.89 mmu). 1H and 13C NMR data, see Table 1. HMBC correlations (DMSO-d6) H-2/C-1; H-7/C-6, 8; H-8/C-7, 9; H-27/C-26, 28; H-28/C-27, 29; H-29a/C-28, 30, 31; H-29b/C(20) Kiyota, H.; Dixon, D. J.; Luscombe, C. K.; Hettstedt, S.; Ley, S. V. Org. Lett. 2002, 4, 3223-3226. (21) Nishikawa, T. In Guide to Seashore Animals of Japan with Color Plates and Keys, II; Nishimura, S., Ed; Hoiku-sha: Osaka, 1995; pp 573610.

Structure Elucidation of Shishididemniols SCHEME 4 a

a

Reagents and conditions: (a) Cbz-Cl, Et3N, CHCl3/MeOH (3:1), rt, 16 h; (b) 2,2-dimethoxpropane, PPTS, CH2Cl2, rt, 1 h; (c) K2CO3, MeOH, rt, 1.5 h.

28, 30, 31; H-1′a/C-1, 2′, 3′; H-1′b/C-1, 2′, 3′; H-2′/C-3′, 4′, 8′; H-4′, 8′/C-2′, 4′, 5′, 6′, 7′, 8′; H-5′, 7′/C-3′, 5′, 6′, 7′; H-9′a/C-6′, 10′, 11′; H-9′b/C-6′, 10′, 11′; H-10′/C-9′, 11′, 12′; H-13′/C-12′, 14′; H-14′/C-12′, 13′; NH-1′/C-1, 1′; OH-2′/C-1′, 2′, 3′; NH-10′/C-10′, 11′, 12′. Shishididemniol B (2). Colorless oil; [R]20.5D -19.4 (c 1.00, MeOH); UV (MeOH) λmax 275 nm ( 1222), 281 (1043); IR (film) νmax 3429, 1642 cm-1; HRESIMS m/z 876.57161 (M + H)+ (calcd for C45H8335ClN3O11, ∆ -1.21 mmu). 1H and 13C NMR data, see Table 1. HMBC correlations (DMSO-d6) H-2/C-1, 3, 4; H-8/C-7, 9; H-27/C-26, 28; H-28/C-26, 27, 29; H-29a/C-28, 30, 31; H-29b/ C-28, 30, 31; H-30/C-29, 31; H-31a/C-29, 30; H-1′a/C-1, 2′, 3′; H-1′b/C-1, 2′, 3′; H-2′/C-1′, 3′, 4′, 8′; H-4′, 8′/C-2′, 4′, 5′, 6′, 7′, 8′; H-5′, H-8′/C-3′, 4′, 5′, 6′, 7′, 8′; H-9′a/C-6′, 10′, 11′; H-9′b/C6′, 10′, 11′; H-10′/C-9′, 11′, 12′; H-11′a/C-9′, 10′; H-11′b/C-9′, 10′; H-13′/C-12′, 14′; H-14′/C-12′, 13′. Preparation of N-Cbz Derivative 3. To a solution of 1 (50 mg, 0.06 mmol) in CHCl3/MeOH (3:1, 0.4 mL) were added triethylamine (66.4 µL) and Cbz-Cl (17 µL). After stirring for 16 h at room temperature, the reaction mixture was evaporated, suspended in H2O, and extracted with EtOAc to give 3 (45 mg, 77.1%): ESIMS m/z 974.37505 (M + H)+. 1H NMR (600 MHz, CD3OD) 7.35-7.27 (m, 5H), 7.29 (d, J ) 8.7, 2H), 6.93 (d, J ) 8.7, 2H), 5.09 (d, J ) -12.3, 1H), 5.06 (d, J ) -12.3, 1H), 4.71 (dd, J ) 7.7, 4.6, 1H), 4.22 (m, 1H), 4.05 (dd, J ) 4.8, 3.3, 2H), 3.99 (m, 1H), 3.78 (m, 1H), 3.69 (d, J ) 6.0, 2H), 3.57 (m, 2H), 3.51-3.40 (m, 6H), 3.39-3.33 (m, 2H), 2.98 (ddd, J ) 7.8, 4.4, 4.4 1H), 2.82 (dt, J ) 4.4, 8.5, 1H), 2.24 (q, J ) 7.8, 2H), 1.73-1.25 (m, 42H), 1.12 (t, J ) 7.8, 3H). Preparation of Bromohydrin 4. To a solution of 3 (40 mg) in dry Et2O (2 mL) was added MgBr2‚OEt2 (90 mg). The solution was stirred at room temperature for 4 h and was then evaporated. The residue was separated by ODS HPLC (gradient elution of 5060% aqueous MeCN) to afford 4 (21 mg): ESIMS m/z 1054.38330 and 1056.35926 (M + H)+. 1H NMR (600 MHz, CD3OD) 7.367.27 (m, 5H), 7.29 (d, J ) 8.3, 2H), 6.93 (d, J ) 8.3, 2H), 5.08 (d, J ) -12.9, 1H), 5.05 (d, J ) -12.9, 1H), 4.71 (dd, J ) 7.6, 4.9, 1H), 4.23 (m, 1H), 4.12 (m, 1H), 4.05 (dd, J ) 5.5, 1.8, 2H), 4.00 (m, 1H), 3.77 (m, 1H), 3.69 (d, J ) 5.5, 2H), 3.67 (m, 1H), 3.57 (m, 2H), 3.52-3.40 (m, 6H), 3.38-3.31 (m, 2H), 2.24 (q, J ) 7.6, 2H), 1.94-1.25 (m, 42H), 1.12 (t, J ) 7.6, 3H).

Preparation of Acetonide 5. To the mixture of 4 (10 mg) and PPTS (pyridinium p-toluenesulfonate) (2.4 mg) in CH2Cl2 (100 µL) was added 2,2-dimethoxypropane (20 µL). The reaction mixture was stirred at room temperature for 1 h and was then evaporated. The residue was partition between EtOAc and H2O. The organic layer was purified by ODS HPLC (gradient elution of 80-100% aqueous MeOH) to give 5 (6 mg): ESIMS m/z 1094.33124 and 1096.36362 (M + H)+. 1H NMR (600 MHz, CD3OD) 7.39-7.25 (m, 5H), 7.30 (d, J ) 8.7, 2H), 6.93 (d, J ) 8.7, 2H), 5.08 (d, J ) -12.6, 1H), 5.06 (d, J ) -12.6, 1H), 4.70 (dd, J ) 7.8, 4.6, 1H), 4.22 (m, 1H), 4.07 (br, 1H), 4.05 (dd, J ) 5.5, 2.7, 2H), 3.99 (m, 2H), 3.78 (m, 1H), 3.69 (d, J ) 5.5, 2H), 3.64 (dd, J ) 7.7, 2.7, 1H), 3.57 (m, 2H), 3.52-3.40 (m, 6H), 3.35 (m, 1H), 2.24 (q, J ) 7.5, 2H), 1.90-1.24 (m, 42H), 1.40 (s, 3H), 1.37 (s, 3H), 1.12 (t, J ) 7.5, 3H). Methanolysis of 1. A 60 mg portion of 1 was dissolved in 5 N HCl/MeOH (1:4), the solution was heated at 100 °C for 4 h, and the reaction mixture was cooled and dried. The residue was separated by ODS flash chromatography with H2O, 20%, 60%, 80% and MeOH to give the mixture of 6 and 6a (11.1 mg). The 1H NMR data of 6a was identical with that of 6 (vide infra). Hydrazinolysis of 1. A 20 mg portion of 1 was dissolved in hydrazine (100 µL), the solution was heated at 120 °C for 15 h, and the reaction mixture was cooled and freeze-dried. The residue was separated by ODS flash chromatography with 20% aqueous MeOH and MeOH to give crude 6 (5.3 mg): 1H NMR (600 MHz, CD3OD) 7.34 (d, J ) 8.7, 2H), 7.07 (d, J ) 8.7, 2H), 4.41 (dd, J ) 8.0, 5.7, 1H), 4.27 (dd, J ) -10.1, 4.2, 1H), 2.52 (dd, J ) -10.1, 7.1, 1H), 3.88 (dd, J ) -11.5, 4.6, 1H), 3.81 (dd, J ) -11.5, 6.0, 1H), 3.66 (m, 1H), 3.06 (d, 7.8, 2H). Preparation of (R)- and (S)-MTPA Derivatives of 6 (10a and 10b). To a solution of crude 6 (1.5 mg) in pyridine (30 µL) was added (S)-MTPACl (20 µL). The residue was partitioned between EtOAc and H2O with 0.1 M Na2CO3.The organic layer was purified by ODS HPLC (gradient elution of 75-100% aqueous MeOH) to yield (R)-MTPA derivative 10a (0.3 mg). The (S)-MTPA derivative 10b (0.5 mg) was prepared in the same way. 10a: 1H NMR (600 MHz, CD3OD) 7.62-7.20 (m, 20H; aromatic), 7.08 (d, J ) 8.5, 2H; H-4′, H-8′), 6.73 (d, J ) 8.5, 2H; H-3′, H-5′), 6.06 (dd, J ) -9.0, 4.8, 1H; H-2′), 4.62 (m, 1H; H-10′), 4.62 (m, 1H; H-11′a), 4.52 (m, 1H; H-11′b), 4.01 (dd, -10.3, 5.2, 1H; H-9′a), 3.97 (dd, -10.3, 5.9, 1H; H-9′b), 3.74 (m, 1H; H-1′a), 3.54 (m, 1H; H-1′b), J. Org. Chem, Vol. 72, No. 4, 2007 1223

Kobayashi et al. 3.54 (s, 3H; OMe in MTPA), 3.50 (s, 3H; OMe in MTPA), 3.37 (s, 3H; OMe in MTPA), 3.30 (s, 3H; OMe in MTPA). 10b: 1H NMR (600 MHz, CD3OD) 7.64-7.26 (m, 22H; aromatic in MTPA, H-4′, H-8′), 6.87 (d, J ) 8.2, 2H; H-5′, H-7′), 6.13 (t, J ) 6.9, 1H; H-2′), 4.62 (m, 1H; H-10′), 4.58 (m, 1H; H-11′a), 4.49 (dd, J ) -11.0, 6.2, 1H; H-11′b), 4.02 (m, 2H; H2-9′), 3.68 (d, 6.2, 2H; H2-1′), 3.39 (s, 3H; OMe in MTPA), 3.35 (s, 3H; OMe in MTPA), 3.34 (s, 3H; OMe in MTPA), 3.20 (s, 3H; OMe in MTPA). Synthesis of 7. To a solution of N-Boc-L-serine methyl ester (550 mg, 2.5 mmol) and p-toluenesulfonic acid monohydrate (6.5 mg, 0.03 mmol) in dry toluene (6 mL) was added 2,2-dimethoxypropane (0.6 mL). The reaction mixture was refluxed for 30 min with stirring and evaporated. The residue was subjected to silica gel flash chromatography (n-hexane/EtOAc ) 4:1) to yield 3-(1,1dimethylethyl)-4-methyl-S-2,2-dimethyl-3,4-oxazolidinedicarboxylate (556 mg, 2.15 mmol, 85.9% yield). To a solution of 3-(1,1dimethylethyl)-4-methyl-S-2,2-dimethyl-3,4-oxazolidinedicarboxylate (556 mg, 2.15 mmol) in THF/MeOH (95:5, 13.2 mL) was added LiBH4 (93 mg, 4.26 mmol). The reaction mixture was stirred at room temperature for 2 h and then evaporated. The residue was purified by silica gel flash chromatography (n-hexane/EtOAc ) 1:1) to obtain 7 (416 mg, 1.82 mmol, 84.5% yield). 3-(1,1Dimethylethyl)-4-methyl-S-2,2-dimethyl-3,4- oxazolidinedicarboxylate: 1H NMR (600 MHz, CDCl3) 4.46-3.96 (m, 3H; CH2O, CH-N), 3.71 (s, 3H; OMe), 1.66-1.34 (m, 15H; five methyls). 7: 1H NMR (600 MHz, CDCl3) 4.14-3.56 (m, 5H; CH2-O × 2, CH-N), 1.60-1.41 (m, 15H; five methyls). Preparation of 9. To a mixture of 7 (277 mg, 1.20 mmol), phenol (113 mg, 1.20 mmol), and triphenylphosphine (314 mg, 1.20 mmol) in toluene (3 mL) was added diethyl azodicarboxylate (628 µL (40% solution in toluene), 1.21 mmol). The reaction mixture was stirred for 18 h at 80 °C and evaporated. The residue was subjected to silica gel flash chromatography (n-hexane/EtOAc ) 4:1) to yield 8 (226 mg, 0.74 mmol, 61.4% yield). To a solution of 8 150 mg (0.49 mmol) in CH2Cl2 (2 mL) was added TFA (1 mL). The reaction mixture was stirred at room temperature for 2 h and then evaporated to yield 9 as a TFA salt (135 mg, 0.48 mmol, 98.0% yield). 8: 1H NMR (600 MHz, CDCl3) 7.28-6.89 (m, 5H; aromatic), 4.30-3.79 (m, 5H; CH2-O × 2, CH-N), 1.63-1.44 (m, 15H; five methyls). 9: 1H NMR (600 MHz, CD3OD) 7.29 (t, J ) 7.7, 2H; aromatic), 7.01-6.95 (m, 3H; aromatic), 4.22 (dd, J ) -10.5, 3.9, 1H; CH2a-O), 4.16 (dd, J ) -10.5, 6.9, 1H; CH2b-O), 3.88 (dd, J ) -11.6, 4.6, 1H; CH2a-O), 3.81 (dd, J ) -11.6, 5.6, 1H; CH2b-O), 3.63 (m, 1H; CH-N). (R)- and (S)-MTPA Derivatives of 9 (11a and 11b). To a solution of 9 (8 mg) in pyridine (50 µL) was added (S)-MTPACl (40 µL). The reaction mixture was stirred at room temperature for 30 min and then evaporated. The residue was partitioned between EtOAc and H2O with 0.1 M Na2CO3.The organic layer was subjected to silica gel flash chromatography with CHCl3 to yield (R)-MTPA derivative 11a (10.9 mg). The (S)-MTPA derivative 11b (11.0 mg) was prepared in the same way. 11a: 1H NMR (600 MHz, CD3OD) 7.58-6.77 (m, 15H; aromatic), 4.06 (m, 1H; CH2a-OPh), 3.98 (m, 1H; CH2b-OPh), 4.61 (m, 1H; CH-N), 4.64 (m, 1H; CH2aOMTPA), 4.54 (m, 1H; CH2a-OMTPA), 3.53 (s, 3H; OMe in MTPA), 3.49 (s, 3H; OMe, in MTPA). 11b: 1H NMR (600 MHz, CD3OD) 7.58-6.81 (m, 15H; aromatic), 4.00 (m, 2H; CH2-OPh), 4.60 (m, 1H; CH-N), 4.57 (m, 1H; CH2a-OMTPA), 4.47 (m, 1H; CH2a-OMTPA), 3.52 (s, 3H; OMe in MTPA), 3.48 (s, 3H; OMe, in MTPA). Preparation of (R)- and (S)-MTPA Derivatives of (R)-2Amino-1-phenylethanol (12a and 12b). To a solution of the commercially available (R)-2-amino-1-phenylethanol (20 mg) in pyridine (50 µL) was added (S)-MTPACl (60 µL). The reaction mixture was stirred at room temperature for 30 min and then evaporated. The residue was partitioned between EtOAc and H2O with 0.1 M Na2CO3. The organic layer was subjected to silica gel flash chromatography with CHCl3 to yield (R)-MTPA derivative 12a (14 mg). The (S)-MTPA derivative 12b (18 mg) was prepared 1224 J. Org. Chem., Vol. 72, No. 4, 2007

in the same way. 12a: 1H NMR (600 MHz, CD3OD) 7.46-7.14 (m, 15H; aromatic), 6.12 (m, 1H; CH-O), 3.74 (m, 1H; CH2a-N), 3.57 (m, 1H; CH2b-N), 3.38 (s, 3H; OMe in MTPA), 3.30 (s, 3H; OMe, in MTPA). 12b: 1H NMR (600 MHz, CD3OD) 7.44-7.32 (m, 15H; aromatic), 6.17 (m, 1H; CH-O), 3.68(m, 2H; CH2-N), 3.40 (s, 3H; OMe in MTPA), 3.20 (s, 3H; OMe, in MTPA). Preparation of (R)- and (S)-MTPA Derivatives of 1 (13a and 13b). To a solution of 1 (5 mg) in pyridine (50 µL) was added (S)-MTPACl (60 µL). The reaction mixture was stirred at room temperature for 30 min and then evaporated. The residue was partitioned between EtOAc and H2O with 0.1 M Na2CO3.The organic layer was purified by ODS HPLC (gradient elution of 90100% aqueous MeOH) to yield (R)-MTPA derivative 13a (6 mg). The (S)-MTPA derivative 13b (5.3 mg) was prepared in the same way. 13a: 1H NMR (600 MHz, CD3OD) 5.07 (m, 1H; H-2), 1.74 (m, 1H; H2-3a), 1.86 (m, 1H; H2-3b), 1.26 (m, 1H; H2-4a), 1.45 (m, 1H; H2-4b), 1.62 (m, 1H; H2-5a), 1.75 (m, 1H; H2-5b), 5.00 (m, 1H; H-6), 2.90 (dd, J ) 9.0, 4.2, 1H; H-7), 2.98 (m, 1H; H-8), 5.07 (m, 1H; H-16), 1.26 (m, 2H; H2-26), 1.46 (m, 2H; H2-27), 3.33 (m, 2H; H2-28), 3.41 (m, 1H; H2-29a), 3.39 (m, 1H; H2-29b), 4.39 (m, 1H; H-30), 4.50 (dd, J ) -14.4, 7.6, 1H; H2-31a), 4.39 (m, 1H; H2-31b), 3.67 (m, 1H; H2-1′a), 3.56 (dd, J ) -14.4, 6,2, 1H; H2-1′b), 6.01 (t, J ) 6.2, 1H; H-2′), 7.14 (d, J ) 8.9, 2H; H-4′/8′), 6.67 (d, J ) 8.9, 2H; H-5′/7′), 4.11 (dd, J ) -9.6, 8.3, 1H; H2-9′a), 3.97 (dd, J ) -9.6, 5.5, 1H; H2-9′b), 4.76 (m, 1H; H-10′), 4.59 (dd, J ) -11.0, 8.3, 1H; H2-11′a), 3.86 (m, 1H; H211′b), 2.53 (m, 1H, H2-13′a), 2.35 (m, 1H; H2-13′b), 0.95 (t, J ) 7.3, 3H; H3-14′). 13b: 1H NMR (600 MHz, CD3OD) 4.93 (m, 1H; H-2), 1.51 (m, 1H; H2-3a), 1.65 (m, 1H; H2-3b), 1.15 (m, 1H; H24a), 1.31 (m, 1H; H2-4b), 1.46 (m, 1H; H2-5a), 1.58 (m, 1H; H25b), 4.92 (m, 1H; H-6), 2.96 (dd, J ) 9.3, 4.2, 1H; H-7), 3.02 (m, 1H; H-8), 5.08 (m, 1H; H-16), 1.31 (m, 2H; H2-26), 1.52 (m, 2H; H2-27), 3.40 (m, 2H; H2-28), 3.47 (dd, J ) -10.3, 5.5, 1H; H229a), 3.43 (dd, J ) -10.3, 6.2, 1H; H2-29b), 4.39 (m, 1H; H-30), 4.50 (dd, J ) -11.0, 4.1, 1H; H2-31a), 4.34 (dd, J ) -11.0, 6.9, 1H; H2-29a), 3.63 (dd, J ) -14.5, 6,2, 1H; H2-1′a), 3.56 (m, 1H; H2-1′b), 6.04 (t, J ) 6.2, 1H; H-2′), 7.26 (d, J ) 8.9, 2H; H-4′/8′), 6.61 (d, J ) 8.9, 2H; H-5′/7′), 3.93 (dd, J ) -9.3, 6.6, 1H; H29′a), 3.74 (br, 1H; H2-9′b), 4.70 (m, 1H; H-10′), 4.73 (dd, J ) -11.7, 5.5, 1H; H2-11′a), 4.54 (dd, J ) -11.7, 6.2, 1H; H2-11′b), 2.52 (m, 1H, H2-13′a), 2.25 (m, 1H; H2-13′b), 0.97 (t, J ) 7.2, 3H; H3-14′). Preparation of (R)- and (S)-MTPA Derivatives of 5 (14a and 14b). To a solution of 5 (2 mg) in pyridine (50 µL) was added (S)-MTPACl (40 µL). The reaction mixture was stirred at room temperature for 30 min and then evaporated. The residue was partitioned between EtOAc and H2O with 0.1 M Na2CO3. The organic layer was purified by ODS HPLC (gradient elution of 90100% aqueous MeOH) to yield (R)-MTPA derivative 14a (3.3 mg). The (S)-MTPA derivative 14b (3.6 mg) was prepared in the same way. 14a: 1H NMR (600 MHz, CD3OD) 5.08 (m, 1H; H-2), 1.79 (m, 1H; H2-3a), 1.55 (m, 1H; H2-3b), 1.55 (m, 1H; H2-4a), 1.42 (m, 1H; H2-4b), 1.53 (m, 1H; H2-5a), 1.45 (m, 1H; H2-5b), 3.95 (m, 1H; H-6), 3.62 (m, 1H; H-7), 4.02 (m, 1H; H-8), 5.08 (m, 1H; H-16), 3.38 (m, 2H; H2-28), 3.41 (m, 2H; H2-29), 4.06 (m, 1H; H-30), 4.40 (dd, J ) -11.2, 5.0, 1H; H2-31a), 4.34 (dd, J ) -11.2, 7.3, 1H; H2-31b), 3.66 (m, 1H; H2-1′a), 3.59 (m, 1H; H2-1′b), 6.00 (dd, J ) 7.3, 5.5, 1H; H-2′), 7.15 (d, J ) 8.7, 2H; H-4′/8′), 6.68 (d, J ) 8.7, 2H; H-5′/7′), 4.11 (dd, J ) -10.5, 9.0, 1H; H2-9′a), 3.97 (dd, J ) -10.5, 5.5, 1H; H2-9′b), 4.77 (m, 1H; H-10′), 4.60 (dd, J ) -11.0, 8.5, 1H; H2-11′a), 3.84 (br, 1H; H2-11′b), 2.54 (m, 1H, H2-13′a), 2.37 (m, 1H; H2-13′b), 0.96 (t, J ) 7.4, 3H; H314′a), 5.06 (d, J ) -12.4, 1H; CH2 in Cbz), 5.03 (d, J ) -12.4, 1H; CH2 in Cbz), 1.39 (s, 3H; CH3 in acetonide), 1.34 (s, 3H; CH3 in acetonide). 14b: 1H NMR (600 MHz, CD3OD) 5.00 (dd, J ) 8.7, 4.1, 1H; H-2), 1.66 (m, 1H; H2-3a), 1.41 (m, 1H; H2-3b), 1.35 (m, 1H; H2-4a), 1.24 (m, 1H; H2-4b), 1.47 (m, 1H; H2-5a), 1.37 (m, 1H; H2-5b), 3.87 (m, 1H; H-6), 3.58 (m, 1H; H-7), 3.98 (m, 1H; H-8), 5.08 (m, 1H; H-16), 3.38 (t, J ) 6.4, 2H; H2-28), 3.40

Structure Elucidation of Shishididemniols (m, 2H; H2-29), 4.05 (m, 1H; H-30), 4.42 (dd, J ) -11.0, 5.0, 1H; H2-31a), 4.34 (dd, J ) -11.0, 6.9, 1H; H2-31b), 3.68 (dd, J ) -14.0, 6.2, 1H; H2-1′a), 3.57 (m, 1H; H2-1′b), 6.05 (dd, J ) 7.3, 6.5, 1H; H-2′), 7.27 (d, J ) 8.7, 2H; H-4′/8′), 6.62 (d, J ) 8.7, 2H; H-5′/7′), 3.93 (dd, J ) -11.6, 6.4, 1H; H2-9′a), 3.75 (br, 1H; H29′b), 4.70 (m, 1H; H-10′), 4.74 (dd, J ) -11.5, 5.5, 1H; H2-11′a), 3.54 (dd, J ) -11.5, 5.6, 1H; H2-11′b), 2.53 (m, 1H, H2-13′a), 2.26 (m, 1H; H2-13′b), 0.98 (t, J ) 7.3, 3H; H3-14′a), 5.03 (s, 2H; CH2 in Cbz), 1.38 (s, 3H; CH3 in acetonide), 1.33 (s, 3H; CH3 in acetonide). Preparation of 16. A 10 mg portion of 4 was treated with NaIO4 (20 mg) in MeOH/H2O (4:1, 1 mL) at room temperature for 1 h, followed by reduction with NaBH4 (23 mg). The reaction mixture was evaporated, and the residue was purified by ODS HPLC (gradient elution of 80-100% aqueous MeOH) to obtain 15 (5.5 mg). To a solution of 15 (4 mg) in pyridine (50 µL) was added tert-butyldiphenylchlorosilane (TBDPSCl) (10 µL). After stirring at room temperature overnight, the reaction mixture was evaporated, suspended in H2O, and extracted with EtOAc. The organic layer was evaporated, and the residue was separated by silica gel flash chromatography (n-hexane/EtOAc ) 4:1) to yield 16 (4.6 mg). 15: ESIMS m/z 644.22893 and 646.23454 (M + H)+. 1H NMR (600 MHz, CDCl3) 7.36-7.28 (m, 5H), 5.43 (m, 1H), 5.09 (s, 2H), 4.12 (m, 1H), 3.85-3.53 (m, 8H), 3.40 (t, J ) 6.7, 2H), 1.851.20 (m, 36H). 16: ESIMS m/z 1120.39025 and 1122.40749 (M + H)+. 1H NMR (600 MHz, benzene-d6) 7.80-7.72 (m, 5H), 7.267.20 (m, 16H), 5.10 (s, 2H), 5.03 (d, J ) 8.7, 1H), 4.20 (m, 1H), 3.97-3.85 (m, 3H), 3.83 (dd, J ) -10.5, 6.0, 1H), 3.74 (dd, J ) -9.4, 6.4, 1H), 3.57 (dd, J ) -8.6, 3.9, 1H), 3.46 (m, 1H), 3.37 (dd, J ) -8.6, 5.5, 1H), 3.20 (t, J ) 6.4, 2H), 1.86-1.17 (m, 36H), 1.20 (s, 9H), 1.14 (s, 9H). Preparation of (R)- and (S)-2NMA Derivatives (17a and 17b). To a solution of 16 (1.5 mg) in CH2Cl2 (300 µL) were added (R)2-naphthylmethoxyacetic acid ((R)-2NMA), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (5 mg), DMAP (0.5 mg), and DMAP‚HCl (0.5 mg), and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated and partitioned between EtOAc and H2O. The organic layer was subjected to silica gel flash chromatography (n-hexane/EtOAc ) 9:1) to obtain the (R)-2NMA derivative 17a (1.5 mg). The (S)2NMA derivative 17b (1.5 mg) was prepared in the same way. 17a: 1H NMR (600 MHz, C6D6) 8.67-7.00 (m, 32H, aromatic), 5.37 (s, 1H, -CH-O in 2NMA), 5.09 (s, 2H; -CH2- in Cbz), 5.08 (m, 1H; H-16), 5.01 (d, J ) 8.7, 1H; NH-30), 4.20 (m, 1H; H-30), 3.95 (m, 1H; H-8), 3.92 (m, 1H; H-7a), 3.89 (m, 1H; H-31a), 3.85 (dd, J ) -10.6, 5.4, 1H; H-7b), 3.73 (dd, J ) -9.6, 6.8, 1H; H-31b), 3.57 (br, 1H; H-29a), 3.36 (br, 1H; H-29b), 3.28 (s, 3H; OMe in 2NMA), 3.20 (m, 2H; H2-28), 1.79 (m, 1H; H-9a), 1.72 (m, 1H; H-9b), 1.46 (m, 1H; H-10a), 1.29 (m, 1H; H-10b), 1.550.80 (m, 32H; H2-11 to H2-15, H2-17-H2-27), 1.19 (s, 9H; tertbutyl), 1.13 (s, 9H; tert-butyl). 17b: 1H NMR (600 MHz, C6D6) 8.03-7.02 (m, 32H, aromatic), 5.12 (m, 1H; H-16), 5.09 (s, 2H; -CH2- in Cbz), 5.01 (d, J ) 8.7, 1H; NH-30), 4.92 (s, 1H, -CH-O in 2NMA), 4.20 (m, 1H; H-30), 3.92 (m, 1H; H-8), 3.90 (m, 1H; H-7a), 3.89 (m, 1H; H-31a), 3.83 (dd, J ) -10.6, 5.4, 1H; H-7b), 3.73 (dd, J ) -9.6, 6.8, 1H; H-31b), 3.57 (br, 1H; H-29a), 3.36 (br, 1H; H-29b), 3.30 (s, 3H; OMe in 2NMA), 3.20 (m, 2H; H228), 1.72 (m, 1H; H-9a), 1.65 (m, 1H; H-9b), 1.34 (m, 1H; H-10a),

1.17 (m, 1H; H-10b), 1.61-0.78 (m, 32H; H2-11 to H2-15, H2-17H2-27), 1.19 (s, 9H, tert-butyl), 1.13 (s, 9H, tert-butyl). Preparation of N-Cbz Derivative 18. To a solution of 2 (40 mg, 0.046 mmol) in CHCl3/MeOH (3:1, 0.4 mL) were added triethylamine (66.4 µL) and Cbz-Cl (17 µL). After stirring at room temperature for 16 h, the reaction mixture was evaporated, suspended in H2O, and extracted with EtOAc. The organic layer was purified by ODS HPLC (gradient elution of 70-100% aqueous MeOH) to afford 18 (21.7 mg, 46.7%). 18: ESIMS m/z 1010.45044 and 1012.44065 (M + H)+. 1H NMR (600 MHz, CD3OD) 7.377.26 (m, 5H), 7.29 (d, J ) 8.7, 2H), 6.94 (d, J ) 8.3, 2H), 5.08 (d, J ) -12.3, 1H), 5.05 (d, J ) -12.3, 1H), 4.71 (dd, J ) 7.6, 4.8, 1H), 4.23 (m, 1H), 4.05 (dd, J ) 5.5, 2.3, 2H), 4.03-3.99 (m, 2H), 3.78 (m, 1H), 3.70 (d, J ) 5.5, 2H), 3.67 (m 1H), 3.58 (m, 2H), 3.52-3.39 (m, 7H), 3.35 (m, 1H), 2.24 (q, J ) 7.8, 2H), 1.891.25 (m, 42H), 1.12 (t, J ) 7.8, 3H). Preparation of Acetonide 19 from 18. To the mixture of 18 (5 mg) and PPTS (1.7 mg) in CH2Cl2 (50 µL) was added 2,2dimethoxypropane (10 µL). The reaction mixture was stirred at room temperature for 1 h and was then evaporated. The residue was partition between EtOAc and H2O. The organic layer was purified by ODS HPLC (gradient elution of 80-100% aqueous MeOH) to give 19 (5.2 mg): ESIMS m/z 1050.45589 and 1052.50406 (M + H)+. 1H NMR (600 MHz, CD3OD) 7.37-7.26 (m, 5H), 7.30 (d, J ) 8.7, 2H), 6.93 (d, J ) 8.7, 2H), 5.08 (d, J ) -12.3, 1H), 5.06 (d, J ) -12.3, 1H), 4.70 (dd, J ) 7.5, 4.8, 1H), 4.22 (m, 1H), 4.05 (dd, J ) 5.6, 2.7, 2H), 4.04-3.98 (m, 2H), 3.96 (m, 1H), 3.80-3.74 (m, 2H), 3.69 (d, J ) 5.4, 2H), 3.57 (m, 2H), 3.52-3.39 (m, 6H), 3.35 (m, 1H), 2.24 (q, J ) 7.8, 2H), 1.841.25 (m, 42H), 1.38 (s, 3H), 1.37 (s, 3H), 1.12 (t, J ) 7.8, 3H). Preparation of 3 from 18. To a solution of 18 (5 mg) in 500 µL of MeOH was added potassium carbonate (18 mg). The reaction mixture was stirred at room temperature for 1.5 h, quenched with acetic acid, and then evaporated. The residue was purified by ODS HPLC (gradient elution of 50-60% aqueous MeCN) to yield 3 (3.5 mg) whose 1H NMR spectrum and [R]D value (3 from 18: [R]20D -17.2 (c 0.10 MeOH), 3 from 1: [R]20D -17.6 (c 0.10 MeOH)) were indistinguishable from those of 3 prepared from 1.

Acknowledgment. Thanks are due to the crew of R/V Toyoshio-maru for assistance in collecting the tunicate. We thank Prof. Takenori Kusumi, Tokushima University, for supplying both (R)- and (S)-2NMA. We also thank Prof. Hidenori Watanabe, University of Tokyo, for valuable discussion and Kohtaro Okada and Yoshinari Miyata for technical support. This work was partially supported by Grant-in-aids from MEXT (Priority Area 16073207) and JSPS (Area B 16380142). Supporting Information Available: FAB-MS/MS data of 1 and 2; 1H NMR, 13C NMR, COSY, TOCSY, HMBC, and HSQC spectra of 1 and 2 in DMSO-d6; 1H NMR spectra of 3-19; 1H NMR, COSY, and TOCSY spectra of 13a and 13b in CD3OD; 1H NMR and COSY spectra of 14a and 14b in CD3OD; and NOESY spectra of 5 and 19 in CD3OD. This material is available free of charge via the Internet at http://pubs.acs.org. JO062013M

J. Org. Chem, Vol. 72, No. 4, 2007 1225